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2012; doi: 10.1101/cshperspect.a008383Cold Spring Harb Perspect Biol  Panagiota A. Sotiropoulou and Cedric Blanpain Development and Homeostasis of the Skin Epidermis

Subject Collection

http://cshperspectives.cshlp.org/cgi/collection/ For additional articles in this collection, see

Copyright © 2012 Cold Spring Harbor Laboratory Press; all rights reserved

on July 3, 2012 - Published by Cold Spring Harbor Laboratory Press http://cshperspectives.cshlp.org/Downloaded from

Development and Homeostasisof the Skin Epidermis

Panagiota A. Sotiropoulou and Cedric Blanpain

IRIBHM, Universite Libre de Bruxelles, Brussels, Belgium

Correspondence: [email protected]

SUMMARY

The skin epidermis is a stratified epithelium that forms a barrier that protects animals fromdehydration, mechanical stress, and infections. The epidermis encompasses different append-ages, such as the hair follicle (HF), the sebaceous gland (SG), the sweat gland, and the touchdome, that are essential for thermoregulation, sensing the environment, and influencing socialbehavior. The epidermis undergoes a constant turnover and distinct stem cells (SCs) are re-sponsible for the homeostasis of the different epidermal compartments. Deregulation of thesignaling pathways controlling the balance between renewal and differentiation often leads tocancer formation.

Outline

1 Functional anatomy of the skin epidermis

2 Stem cells with different proliferationdynamics regulate epidermal homeostasis

3 Multipotency and plasticity of epidermalstem cells

4 Signaling pathways and gene networkscontrol tissue differentiation in the skin

5 Genomic maintenance in epidermal stem cells

6 Epidermal stem cells and cancer initiation

7 Therapeutic applications of skinstem/progenitor cells

References

Editors: Patrick P.L. Tam, W. James Nelson, and Janet Rossant

Additional Perspectives on Mammalian Development available at www.cshperspectives.org

Copyright # 2012 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a008383

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1 FUNCTIONAL ANATOMY OF THE SKINEPIDERMIS

The skin consists of the epidermis and its underlying der-mis. The epidermis is a stratified epithelium containingone layer of proliferative cells and several layers of differen-tiated cells. The formation of the different layers of theepidermis (stratification) occurs during embryonic devel-opment, so that animals already present a functional barrierat birth. In mice, stratification begins around embryonicday 15 (E15) and coincides with the rotation of the plane ofcell division from parallel to the basal membrane (symmet-ric cell division), which results in tissue expansion, toperpendicular (asymmetric cell division), which results inone basal proliferative and one suprabasal committed cell(Smart 1970). The asymmetric division is driven by thepolarized localization of the proteins LGN and NuMA tothe apical cortex of the dividing basal cells (Lechler andFuchs 2005; Poulson and Lechler 2010). Serum responsefactor is required to establish a rigid cortical actomyosinnetwork, allowing the critical shape changes associatedwith mitosis (Luxenburg et al. 2011). Using in vivo skin-specific RNA interference, it has been recently shown thatNuma1, LGN, and dynactin promote asymmetric divisionsby reorienting the mitotic spindle, and that Notch may beinvolved in this process (Williams et al. 2011), thus con-necting stratification with induction of differentiation.

Hair follicle (HF) morphogenesis relies on a series ofcross talks between the epithelium and its underlying mes-enchyme (Fig. 1). Pioneering reconstitution experimentsshowed that the dermis instructs the generation of HFsfrom hairless regions, demonstrating the critical role ofthe mesenchyme in specifying the location and numberof HFs (Hardy 1992). The morphogenesis of the mouseHF requires at least three signals, the first coming fromthe mesenchyme at E15 and resulting in the formation ofthe hair placode; the second at E16 from the newly formedplacode to the dermal mesenchymal cells, instructing theformation of the dermal papilla (DP); and the third at E18from the DP, stimulating the proliferation and differentia-tion of the HF cells (Fig. 1) (Blanpain and Fuchs 2006).Mature HFs consist of one layer of basal cells called theouter root sheath (ORS) that expresses Keratin-14 (K14)and K5 and contacts the basement membrane. HF progen-itors residing in the lower part of the HF, called the matrixcells, proliferate and differentiate into the six concentric celllayers of differentiated cells (Fig. 1) (Blanpain and Fuchs2006). When HF morphogenesis is completed, the lowertwo-thirds of the HF degenerates by apoptosis in a processcalled catagen, and the permanent portion enters a restingstage (telogen) until a new cycle of hair growth takes place(Blanpain and Fuchs 2006).

The sensory function of the skin is mediated by theactivity of specific receptors on distinct cells residing inthe epidermis such as keratinocytes, Merkel cells (MCs),and free nerve endings (Lumpkin and Caterina 2007). MCsare neuroendocrine cells that can be found in hairy andglabrous skin. They are clustered in touch-sensitive areascalled touch domes (Boulais and Misery 2007). MCs ex-press intermediate filaments of simple epithelia, such asK8, K18, and K20, as well as neuropeptides, proneuraltranscription factors, and components of presynaptic ma-chinery, suggesting that MCs arise from neural crest cells.However, lineage-tracing experiments using epidermal andneural crest–specific promoters unambiguously show thatMCs arise from epidermal cells by a mechanism that re-quires Atoh1 expression (Morrison et al. 2009; Van Key-meulen et al. 2009).

Thermoregulation in the epidermis is controlled main-ly by the sweat glands (Shibasaki et al. 2006). In humans,sweat glands are formed during embryonic developmentand reach the morphology, size, and final number of theadult appendages by the eighth month of gestation (Saga2002). It is still unclear how homeostasis of sweat glands isregulated and how their specification occurs. Lineage-trac-ing studies during embryonic development and adult lifewill clarify the mechanisms that regulate their specificationand homeostasis.

2 STEM CELLS WITH DIFFERENT PROLIFERATIONDYNAMICS REGULATE EPIDERMALHOMEOSTASIS

Homeostasis of the different compartments of the skinepidermis is accomplished by the activity of three differentclasses of stem cells (SCs) that reside in specific niches andexhibit distinct turnover rates (Fig. 2).

2.1 Bulge Stem Cells

The first functional characteristic of bulge SCs was theirrelative quiescence (Cotsarelis et al. 1990). Nucleotide ana-log pulse-chase experiments showed the presence of label-retaining cells (Bickenbach 1981) in the bulge region of theHF (Cotsarelis et al. 1990), indicating that bulge cells aremore quiescent than the other epidermal cells. HF lineage-tracing experiments using tetracycline-regulated greenfluorescent protein (GFP)-tagged histone H2B (Tet-o-H2B-GFP) (Tumbar et al. 2004) or the expression of re-porter genes under the control of HF-specific promoterssuch as K15 (Morris et al. 2004), Lgr5 (Jaks et al. 2008), orK19 (Youssef et al. 2010) showed that the cells residing inthe bulge and upper germ are responsible for HF regener-ation during adult homeostasis. Bulge SCs isolated by

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microdissection (Rochat et al. 1994; Oshima et al. 2001) orby flow cytometry based on the expression of CD34 (Trem-pus et al. 2003; Blanpain et al. 2004) or K15-GFP (Morriset al. 2004) and cultured in vitro produced large prolifera-tive colonies that could be propagated for a long time.Transplantation of the progeny of a single bulge SC cangenerate all HF lineages (Blanpain et al. 2004; Claudinotet al. 2005). During anagen, bulge SCs migrate along theORS to the matrix, where they proliferate, differentiate, and

produce the inner root sheath (IRS) and the hair shaft(Oshima et al. 2001; Ito et al. 2004; Tumbar et al. 2004).

Quantitative H2B-GFP label-retention studies duringnormal hair homeostasis showed that almost all bulge SCsaccomplish at least one division (94%) over a hair cycle,whereas the majority of bulge SCs accomplish three ormore divisions (Waghmare et al. 2008). Bromodeoxyuri-dine pulse during the early stage of HF regeneration indi-cates that the first cells to incorporate bromodeoxyuridine

E14

Dermal cells

Basal layer Suprabasal layer

Dermal condensate

Hair placode

Hair germ

Dermal papilla

Hair matrix

Inner root sheath

Basal layerSpinous layer Granular layerStratum corneum

Arrector pili muscle

Sebaceous gland

Outer root sheath

Hair shaft precursors

Hair shaft

Matrix

Dermal papilla

E15

E16

E17

E18

Inner root sheathCompanion layer

E12 Basal layer

Spinous layer

Granular layer

Signal 1

Signal 2

Signal 3

P4

Basement membrane

Outer root sheath

Figure 1. Skin morphogenesis and generation of epidermal cell lineages. During embryonic development the single-layered epidermis stratifies, resulting in layers of differentiated cells forming the mature epidermis and itsappendages.

Regulation of Skin Stem Cells

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are the cells of the hair germ (HG), followed by bulge SCs,suggesting a two-step mechanism of HF regeneration inwhich the first cells to be activated are the HG cells, fol-lowed by bulge SCs (Greco et al. 2009). After an initial burstof proliferation that accompanies the early stage of HFregeneration, bulge SCs continue to divide at a lower rateduring all of the growing stages of the hair cycle and thenstop dividing during catagen (Sotiropoulou et al. 2008;Waghmare et al. 2008). Lineage tracing of single bulgeSCs shows their ability to migrate from the bulge to theHG and their multipotency at a clonal level (Zhang et al.2009b). Although the vast majority (.90%) of Lgr5+ cellsexpress CD34 and are quiescent like all other bulge cells,Lgr5+ cells in the HG have been proposed to cycle frequently(Jaks et al. 2008). However, H2B-GFP label-retention stud-ies show that both bulge and HG contain cells that did notcycle extensively during the previous haircycle (Nowaket al.2008; Zhang et al. 2009b; Hsu et al. 2011). Defining moreprecisely the respective contribution of HG versus bulge SCsduring HF regeneration will require the establishment ofnew inducible CRE mice because all the current modelsare expressed in both the bulge and HG areas.

Interestingly, although the existence of a morphologi-cally distinct bulge region (Fig. 2) and the expression of

bulge SC markers begin during the first hair cycle in 3-week-old mice (Blanpain et al. 2004), the slow-cyclingproperties of the prospective bulge cells are acquired rela-tively early during HF morphogenesis. H2B-GFP pulse-chase experiments starting at E18.5 show that the slow-cycling property of some cells of the upper part of the HFis acquired during the first days of postnatal life, and at theend of morphogenesis when the bulge SC niche is formed,and that all label-retaining cells are located in bulge andHG regions (Nowak et al. 2008). The specification of theprospective slow-cycling bulge SC depends on Sox9 expres-sion, as early follicle progenitors fail to sustain hair growthand to establish the adult bulge SC niche in the absence ofSox9 (Vidal et al. 2005; Nowak et al. 2008).

During HF regeneration, some bulge SCs migrate outand home back to their niche after accomplishing severalcell divisions. Some of these cells can directly contribute tothe next round of HF regeneration as normal resident bulgeSCs, whereas others that pass a certain threshold of com-mitment are still able to home back to their niche but areunable to proliferate anymore. In contrast, these cells con-tribute to the establishment of their niche by providing aphysical anchor between the HF SC and the old club hairand secrete high levels of bone morphogenetic protein-6

IFE

SG

Multipotent bulge SC

Isthmus-resident SC

Interfollicularepidermis-resident SC

Sebaceousgland-resident SC

Figure 2. Different types of SCs regulate epidermal homeostasis. Lineage-tracing analysis provided evidence for thecontribution of each type of SC of the epidermis during normal homeostasis. (Middle boxed image reproduced, withpermission, from Snippert et al. 2010 # Science.)

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(BMP6) and fibroblast growth factor-18 (FGF18) (Hsu et al.2011) that regulate SC quiescence (Blanpain et al. 2004).

2.2 Stem Cells of the Infundibulumand the Sebaceous Gland

The homeostasis of the upper part of the HF and the seba-ceous gland (SG) is maintained independently of bulge SCsand relies on the presence of unipotent SCs (Fig. 2) char-acterized by the expression of the cell surface markersMTS24, Lrig1, Blimp1, or Lgr6, which are able to differ-entiate into all epidermal lineages upon transplantationinto immunodeficient mice (Horsley et al. 2006; Nijhofet al. 2006; Jensen et al. 2008, 2009; Snippert et al. 2010).Lgr6 lineage-tracing experiments showed that these cellscontribute to SG homeostasis but also mark some cells inthe interfollicular epidermis (IFE) (Snippert et al. 2010). Itis still not clear whether Lgr6 marks bipotent SCs or twodifferent SC populations (Blanpain 2010).

2.3 Stem Cells of the Interfollicular Epidermis

The IFE was initially believed to be maintained by smallunits of proliferation containing one quiescent SC and sev-eral transit-amplifying cells (Potten 1974, 1981) that wouldamplify the number of differentiated cells and therebylimit the number of cell divisions that a SC needs to ac-complish to maintain epidermal homeostasis (Jones et al.2007). Retroviral marking of basal IFE cells and their prog-eny revealed the presence of columns of marked cells fromthe basal layer to the top of the cornified layer, which sup-ports the notion that epidermal proliferative units (EPUs)do exist in vivo (Fig. 2) (Kolodka et al. 1998; Ghazizadehand Taichman 2001, 2005). Subsequent studies usingmutagens to restore the open reading frame of the stop-enhanced GFP reporter suggested that EPUs do not formthe hexagonal shape predicted by histological studies andthat SCs can migrate between EPUs (Ro and Rannala 2004).Using clonal analysis of the tail epidermis coupled withmathematical modeling, Clayton and colleagues showedthat the number of labeled clones decreases with timetogether with an ever-increasing size of the labeled clones.The absence of steady state in the number and size oflabeled clones is not expected based on the SC–transit-amplifying cell model of EPU. These data are instead com-patible with a model in which the basal epidermis containsonly one type of progenitors, without the involvement oftransit-amplifying cells, which choose randomly to renewor differentiate with a defined probability (Clayton et al.2007).

To maintain the tissue with a constant cell number, thefate of each cell depends on the division rate and the

proportion of cells that differentiate thereafter, and thereshould be an exquisite balance between the number of cellslost by desquamation and cells that proliferate at a giventime point. According to this model, epidermal homeostasisis maintained by a combination of symmetric and asymmet-ric cell divisions; the majority of divisions are asymmetricand progenitors divide every 6–7 d. Using the same strategyof clonal analysis, it has been proposed that the ear epi-dermis is also maintained by one type of progenitor thatrandomly chooses between two fates, although the param-eters of the cell cycle and the probability to self-renew anddifferentiate were different (Doupe et al. 2010). Althoughthese data challenge our current view of hierarchical homeo-stasis, it remains to be shown that the cells targeted in thesestudies are not a particular subpopulation of epidermalcells.

In conclusion, these data indicate that under physio-logical conditions, the skin epidermis is maintained bydifferent SC populations (Fig. 2). New tools such as H2B-GFP label retention and clonal analysis have been instru-mental in challenging our current view of SC dynamics inthe skin epidermis. A lot of questions still remain unan-swered. What is the exact contribution of bulge SC versusHG cell in the initiation of HF regeneration? What is theproportion of multipotent SCs in the bulge and the HG?Do HF SCs participate in IFE homeostasis? Do bulge SCsdivide randomly and compete for the niche? Does IFEcontain slow-cycling cells?

3 MULTIPOTENCY AND PLASTICITY OFEPIDERMAL STEM CELLS

The notion that the skin epidermis contains multipotentSCs with the ability to differentiate into all epidermal celllineages including the IFE, the SG, and all HF lineages camefrom transplantation studies, in which different types ofepidermal cells were transplanted together with embryonicskin mesenchyme onto immunodeficient mice. Pioneeringstudies were performed by the group of Barrandon, inwhich different regions of the epidermis were microdis-sected and transplanted into immunodeficient mice. Thesestudies demonstrated the ability of bulge SCs to re-form allepidermal lineages (Rochat et al. 1994; Oshima et al. 2001)and suggested the existence of multipotent bulge SCs. Sim-ilarly, transplantation of fluorescence-activated cell sorting(FACS)-isolated bulge SCs together with embryonic mes-enchyme into immunodeficient mice also resulted in epi-dermal regeneration and differentiation into the completerepertoire of epidermal cell types (Morris et al. 2004).Transplantation of the progeny of a single cultured bulgeSC also led to differentiation into all epidermal lineagesincluding bulge SCs that were able to renew and cycle for

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more than a year after transplantation (Blanpain et al.2004) and to be serially transplanted (Claudinot et al.2005), confirming the enormous renewal and differentia-tion potential of bulge SCs.

Lineage tracing using inducible CRE specific for adultbulge SCs or constitutive Sonic hedgehog (Shh)-CRE,which marked embryonic HF progenitors and all futureadult hair cells, showed that during homeostasis the IFEis maintained independently of the HF SCs and duringhomeostasis bulge SCs essentially contribute to HF regen-eration (Morris et al. 2004; Levy et al. 2005). However,following wounding, bulge SCs rapidly migrate towardthe injured region (Tumbar et al. 2004) and actively par-ticipate in the repair of the skin barrier (Fig. 3) (Ito et al.2005; Levy et al. 2007). However, there are conflicting re-sults regarding the long-term fate of the bulge SCs thatmigrated into the IFE. Some studies suggest that they areonly transiently present, whereas others report that theycan be maintained long term in the regenerated epidermis(Ito et al. 2005; Levy et al. 2007; Nowak et al. 2008).

The contribution of bulge SCs to SG homeostasis is notentirely clear. Although SG arises during morphogenesisfrom HF progenitors that may be common to bulge SCs, itis unclear whether bulge SCs participate in the normalrenewal of the gland in the absence of pathological condi-tions. Lineage tracing using K15CREPR mice showed la-beling of rare SG cells (Morris et al. 2004), whereas Lgr5(Jaks et al. 2008) and K19CREER failed to label the SGduring homeostasis (Youssef et al. 2010; Lapouge et al.2011). In contrast, during pathological conditions thatstimulate SG proliferation such as oncogenic Ras expres-sion (Lapouge et al. 2011) or Blimp1 deletion (Horsleyet al. 2006), bulge SCs can contribute to the expansion ofSG. Under physiological conditions, the SG contains itsown unipotent progenitors that ensure its high cellularturnover. One population of cells expressing MTS24,

Lrig1, and Lgr6 located in the upper isthmus and the lowerinfundibulum has extensive renewal capacities in cultureand is capable of differentiating into all epidermal lineagesupon transplantation (Nijhof et al. 2006; Jensen et al. 2008,2009; Snippert et al. 2010). Lineage tracing using Lgr6 pro-moter confirmed the existence of unipotent SG progenitorcells in the isthmus region that ensure SG homeostasis andthat are also capable of migrating toward the IFE to partic-ipate in wound repair (Snippert et al. 2010).

Although the IFE during homeostasis clearly containsunipotent progenitors giving rise to suprabasal differenti-ated cells, overexpression of a constitutively active form ofb-catenin in adult IFE progenitors induces de novo HFmorphogenesis that recapitulates all aspects of normal em-bryonic HF development including the formation of DP(Gat et al. 1998). Not all epidermal compartments areequally sensitive to b-catenin. Infundibulum and seba-ceous progenitors are the most sensitive epidermal cellsand bulge SCs are the most resistant (Baker et al. 2010).De novo HF morphogenesis has been reported to occurduring wound healing, and K15CREPR lineage tracingsuggested that new follicles arise from IFE, isthmus, orinfundibulum cells rather than bulge SCs. Interestingly,b-catenin is also required for HF morphogenesis duringwound healing, and increasing the level of Wnt ligandspromotes this process (Ito et al. 2007).

In conclusion, upon wounding, epidermal SCs rapidlyrespond to changes in their microenvironment and adopt adifferent fate than during normal homeostasis (Fig. 3).These data also indicate that transplantation experimentsinto immunodeficient mice, although very informativeabout the differentiation potential of SCs, mimic awounding environment and extrapolation of the resultsof such experiments to what happens during physiologicalconditions can be misleading. They also stress the impor-tance of the underlying mesenchyme in dictating the

EpIn

In

W

W

BM

Bu

LowerORS

Bu

DP

4 h 24 h 24 h

Figure 3. Multipotency of bulge SCs during wound healing. Upon IFE wounding, HF bulge SCs migrate to the site ofwounding and contribute to the regeneration of IFE. (Bottom images reproduced, with permission, from Tumbaret al. 2004 # AAAS.)

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differentiation potential of epithelial SCs. Nonhairy epithe-lia including central corneal cells (Ferraris et al. 2000) andepithelial thymic cells (Bonfanti et al. 2010) can adopt HFfate when grafted together with embryonic backskin der-mis. More studies will be needed to identify the inductivefactors secreted by the mesenchyme and to determine themolecular mechanisms of their cellular memory and epi-thelial plasticity.

4 SIGNALING PATHWAYS AND GENENETWORKS CONTROL TISSUEDIFFERENTIATION IN THE SKIN

4.1 Epidermal Stratification, Renewal,and Differentiation

Although the precise mechanisms controlling epidermalstratification, homeostasis, and renewal upon injury arestill unfolding, a number of molecules crucial for theseprocesses have been identified. The first transcription fac-tor specifically expressed in the epidermis is p63, a memberof p53 family that consists of two major isoforms, TAp63and DNp63, which express and lack the N-terminal trans-activation domain, respectively. Mice lacking p63 exhibitmajor defects in all stratified epithelia including the epi-dermis, and die immediately after birth because of theabsence of skin barrier (Mills et al. 1999; Yang et al. 1999;Koster and Roop 2007). It is a matter of intense debatewhether p63 regulates the specification of stratified epider-mis (Mills et al. 1999), or controls the renewal of epidermalprogenitors (Yang et al. 1999), or a combination of bothmechanisms. Forced expression of TAp63 in the lung epi-thelium induces stratification and keratinization, support-ing its role in the induction of stratification (Koster et al.2004). A more recent study suggests that p63 is essential forthe maintenance of the proliferative ability of SCs, whereasit is dispensable for their initial commitment and differen-tiation (Senoo et al. 2007). Several p63 target genes havebeen identified, including Perp (Ihrie et al. 2005), Fras1(Koster and Roop 2007), and the basal integrins b1 andb4 (Carroll et al. 2006).

The mitogen-activated protein kinase (MAPK) cascadeis a critical regulator of the balance between epidermalproliferation and differentiation. Deletion of Mek1/2 re-sults in decreased proliferation, leading to epidermal hy-poplasia (Scholl et al. 2007). Simultaneous deletion ofErk1/2 also results in proliferation defects and hypoplasia(Dumesic et al. 2009). Conversely, activation of Ras, Raf,or the downstream Mek1 in the skin results in hyperprolif-eration accompanied by epidermal hyperplasia and down-regulation of differentiation markers (Tarutani et al. 2003;Scholl et al. 2004). Activation of the MAPK pathway dur-ing normal homeostasis is mediated by tyrosine kinase

receptors such as epidermal growth factor receptor (EGFR)and by basal integrins, and is negatively regulated by adhe-sion molecules. Accordingly, reduced EGF signaling leadsto a decrease in IFE proliferation, whereas constitutive ac-tivation of EGFR results in hyperproliferation and epider-mal tumors (Janes and Watt 2006). Human IFE SCs expresshigh levels of b1-integrins (Jones and Watt 1993; Joneset al. 1995), whose overexpression leads to their prolifera-tion and expansion (Zhu et al. 1999; Haase et al. 2001). Onthe contrary, deletion of the cytoplasmic proteins associat-ed with cadherin cell–cell adhesions, a- or p120-catenin,leads to hyperproliferative epidermis because of sustainedactivation of the Ras-MAPK pathway (Vasioukhin et al.2001; Perez-Moreno et al. 2006). Consistent with its roleas a regulator of proliferation versus differentiation in theepidermis, MAPK signaling is crucial for epidermal tumorformation, and indeed most squamous cell carcinomas(SCCs) are characterized by activation of the Ras pathwayand up-regulation of MAPK activity (Khavari and Rinn2007).

The canonical Notch pathway is a major regulator ofIFE differentiation. Notch receptors are expressed in thesuprabasal committed cells of the IFE, whereas their ligandJagged2 is expressed in the basal layer (Watt et al. 2008).Monitoring the activation of the Notch pathway using anantibody against the active form of Notch, lineage-tracingexperiments using Cre recombinase triggered by the acti-vation of Notch1, or expression of Notch target gene Hes1revealed that the Notch pathway is active mainly in supra-basal cells (Blanpain et al. 2006; Vooijs et al. 2007; Mori-yama et al. 2008). Complete inactivation of the Notchpathway in mice deficient for g-secretase 1 and 2 or mul-tiple Notch receptors (Pan et al. 2004) or the transcriptionfactor that relays Notch signaling (RBP-Jk) (Blanpain et al.2006) or Hes1 (Moriyama et al. 2008) leads to a lack ofspinous differentiation and loss of skin barrier formation.Conversely, overexpression of Notch intracellular domainin the epidermis leads to spinous layer expansion (Uytten-daele et al. 2004; Blanpain et al. 2006). Collectively, thosestudies show that the Notch pathway regulates the earlysteps of IFE commitment and differentiation. Notch actssynergistically with the AP2 protein family to regulate theexpression of C/EBP transcription factors, which in turninfluences terminal differentiation and skin barrier forma-tion (Wang et al. 2008). Accordingly, AP2- (Wang et al.2008) or C/EBP-deficient (Lopez et al. 2009) mice exhibitdefects in epidermal differentiation.

Whereas Notch represents a proto-oncogene in manymammalian tissues, in the skin epidermis, consistent withits role in promoting terminal differentiation, Notch sig-naling acts as a tumor suppressor, and several mouse mod-els deficient for factors of the Notch pathway develop

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tumors and are more susceptible to cancer developmentupon Ras activation (Nicolas et al. 2003; Proweller et al.2006). In vitro studies using human keratinocytes haveindicated that Notch1 acts as a tumor suppressor gene byup-regulating p21 (Rangarajan et al. 2001). However, invivo studies indicated that Notch1 promotes tumorigene-sis in a non-cell-autonomous manner by creating a wound-like microenvironment because of persistent skin barrierdefects (Blanpain et al. 2006; Demehri et al. 2009). In ad-dition to cancer, Notch signaling controls inflammatoryresponses. Notch deficiency during embryonic develop-ment leads to non-cell-autonomous B-cell lymphoprolifer-ative disorder, whereas in adult mice it results in severeatopic dermatitis because of elevated levels of thymic stro-mal lymphopoietin, possibly because of impaired barrierformation, consistent with the finding that patients suffer-ing from atopic dermatitis have reduced Notch expressionin the skin (Dumortier et al. 2010). The elevated levels ofthymic stromal lymphopoietin may subsequently act sys-temically and lead to allergic asthma development, explain-ing the elevated cases of asthma within atopic dermatitispatients (Demehri et al. 2009).

In addition to these signaling pathways, other genescontrol later steps of IFE differentiation. Mice deficientfor Irf6 exhibit hyperproliferative epidermis with concom-itant lack of differentiation (Ingraham et al. 2006; Richard-son et al. 2006). Mice lacking Klf4 die perinatally bydehydration because of skin barrier loss, associated withalterations in late-stage differentiation markers, such as thecornified envelope gene Sprr2a (Segre et al. 1999). Con-versely, ectopic expression of Klf4 in the basal layer leads toa premature barrier formation, accelerated differentiation,and reduced proliferation (Jaubert et al. 2003). Grhl3, oneof the mammalian homologs of the Drosophila transcrip-tion factor grainyhead, is important for the establishmentof the skin barrier function. Grhl3 is expressed in the em-bryonic epidermis from E8.5 to E17.5 and is maintained atlow levels thereafter (Auden et al. 2006). Grhl3-null miceexhibit defective skin barrier and wound repair because ofdefective protein cross-linking in the suprabasal layers ofthe epidermis mediated by the reduced transglutaminase-1expression (Ting et al. 2005; Boglev et al. 2011). These dataindicate that different signaling pathways and transcriptionfactors orchestrate the balance between renewal and differ-entiation of the skin epidermis.

4.2 HF Morphogenesis and Cycling

Wnt/b-catenin signaling is the earliest signaling pathwaycontrolling HF specification. Deletion of b-catenin in theskin epidermis or overexpression of Dkk1, a soluble inhib-itor of Wnt signaling, results in the absence of HF with no

sign of HF or DP formation (Huelsken et al. 2001; Andlet al. 2002). Conditional deletion of b-catenin in adultmouse epidermis leads to HF degeneration and cyst forma-tion, demonstrating the essential function of b-catenin inmaintaining the identity of adult HF SCs (Lowry et al.2005). Increased levels of b-catenin in adult HF SCs resultsin the shortening of telogen and the acceleration of telogento anagen transition (Van Mater et al. 2003; Lo Celso et al.2004; Lowry et al. 2005), with a concomitant increase inbulge SC proliferation (Lowry et al. 2005). Transcriptionalprofiling of bulge SCs during telogen and anagen, as well asfollowing b-catenin gain-of-function expression, identi-fied a set of genes modulated by Wnt/b-catenin signalingand associated with anagen reentry. Chromatin immuno-precipitation and functional studies identified amongthem CyclinD1, TIMP3, and biglycan as direct b-catenintarget genes (Lowry et al. 2005). Tcf3 and Tcf4 are alsoexpressed in early undifferentiated embryonic epidermalprogenitors and in adult HF SCs and their progeny, andseem to act at least partially as transcriptional repressors ina Wnt-dependent and -independent manner. Their simul-taneous deletion leads to rapid HF disappearance (Ngu-yen et al. 2009). b-Catenin signaling acts upstream of theEda-A1/Edar/NF-kB signaling pathway, which controlsHF morphogenesis during the early stage of HF specifica-tion, but EDA/EDAR/NF-kB activity helps to sustain Wntactivity at latter stages (Zhang et al. 2009a). Although over-expression of b-catenin during adult homeostasis leads tode novo HF formation from the IFE and SG and to a lesserextent from HF, high levels of b-catenin signaling duringembryonic development lead to expansion of HF placodes,which fail to down-grow and differentiate. Interestingly,increasing b-catenin during embryonic development en-hances innervations and pigmentation, as a consequenceof higher expression of proteins such as KitL and axonalguidance molecules (Zhang et al. 2008). FACS-isolated DPcells rapidly lose their inductive properties upon in vitroculture, which can be rescued by addition of Wnt3a (Ki-shimoto et al. 2000). b-Catenin deletion specifically in DPcells results in a strong decrease in HF progenitor prolifera-tion and HF terminal differentiation, leading to a prematureentry in catagen and preventing bulge SCs from reinitiating anovel cycle of HF regeneration (Enshell-Seijffers et al. 2010).In adult epidermis, b-catenin regulates the expression ofnephronectin by HF SCs, which in turn regulates the attach-ment of the arrector pili muscle to the top of the bulge(Fujiwara et al. 2011). These data nicely illustrate that similarsignals regulate epithelial SCs and the underlying inductivemesenchymal compartments, and that Wnt/b-catenin sig-naling acts as key regulator of the HF fate specification in-trinsically and through the regulation of SC niche. Moreover,deregulation of Wnt/b-catenin signaling leads to HF tumors

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in mice (Gat et al. 1998) and is activated in human HF tu-mors (Chan et al. 1999), suggesting that b-catenin acts as anoncogene in the skin.

Shh is expressed in embryonic epidermis in HF down-growths just after placode progression. Shh is geneticallydownstream of Wnt signaling because Shh is not expressedin the absence ofb-catenin (Huelsken et al. 2001; Andl et al.2002), whereas Lef1 and b-catenin are present in the HG ofShh-null mice (St-Jacques et al. 1998). Shh-CRE marks allHF lineages including the SG, showing that all HF cellsderive from the initial HG (Levy et al. 2005). Shh-nullmice develop placodes and HG, which fail to invade thedermis and to expand into the different HF lineages (St-Jacques et al. 1998), demonstrating the essential role of Shhfor HF progenitor expansion and differentiation. Shh isalso expressed in a cyclic manner during the hair cycle.Shh is expressed in adult HG and then in the prospectiveIRS cells and finally in one polarized side of the matrixcalled the lateral disk, which gives rise preferentially toIRS (Panteleyev et al. 2001). In addition, Shh is expressedin the mesenchymal part of the hair (Oro and Higgins2003), suggesting that Shh signaling plays a role in theepithelial–mesenchymal interaction regulating hair cycle.Ectopic administration of Shh or Shh agonists stimulatesthe proportion of hair in its growing stage (Sato et al. 1999;Paladini et al. 2005), and administration of Shh inhibitorblocks anagen progression (Wang et al. 2000; Silva-Vargaset al. 2005), showing that Shh exhibits a role in mediatinganagen progression during the hair cycle. Gli2-deficientmice present an arrest in HF development similar to Shh-null mice, which is rescued by overexpression of a consti-tutively active but not wild-type form of Gli2 in the epi-dermis (Mill et al. 2003), showing that Gli2 expression inthe epidermis is essential to mediate Shh signaling duringHF morphogenesis. Constitutive activation of Shh leadsto the formation of familial and sporadic basal cell carci-nomas (BCCs) (Epstein 2008), and Shh inhibitors are cur-rently administered in clinical trials for advanced andmetastatic BCC (Von Hoff et al. 2009).

Normal HF development and maintenance requires abalance between BMP ligands (e.g., BMP2 and BMP4) andBMP antagonists (e.g., Noggin). Noggin is expressed by theunderlying mesenchyme, and its deletion results in areduction of HF specification and a decrease in HF matu-ration, partly by a failure to induce Lef1 expression andamplify the Wnt response (Botchkarev et al. 1999; Jamoraet al. 2003). Epidermal cells express Bmpr1a, whose condi-tional deletion results in the formation of large placodelikestructures expressing high levels of Lef1, which continueto grow as a mass of undifferentiated ORS-like cells, failto terminally differentiate, and finally degenerate into hy-perproliferative cystic structures leading to HF tumors

(Kobielak et al. 2003; Andl et al. 2004; Ming Kwan et al.2004; Yuhki et al. 2004). In adult mice, BMP signalingregulates bulge SC quiescence. During the quiescent stageof the hair cycle, BMP2 and BMP4 are highly expressed bydifferent dermal cells including fibrobasts, DP cells, andadipocytes (Plikus et al. 2008). The expression of dermalBMP progressively decreases during the resting stage, pro-moting a switch from quiescence to activation of bulge SCsand the initiation of HF regeneration (Plikus et al. 2008).Bulge SCs secrete high levels of BMP6, which promotestheir quiescence in an autocrine or paracrine manner(Blanpain et al. 2004). BMP signaling controls HF SC qui-escence in part by regulating the expression of Nfatc1,which is abolished in BMPR1a-null epidermis. Loss ofNfatc1 stimulates bulge SC proliferation and anagen pro-gression through loss of CDK4 repression (Horsley et al.2008). Transcriptional profiling showed that DP cells ex-press a number of BMP receptors and several BMPs, themost potent being BMP6, promoting the maintenance ofthe DP-inductive properties upon in vitro culture (Rendlet al. 2005, 2008). Accordingly, conditional ablation ofBMPR1a specifically in DP cells in vivo results in loss ofthe DP-inductive capacity in HF reconstitution assays(Rendl et al. 2008).

During embryonic development, Runx1 is expressed inboth the embryonic HF and its underlying mesenchyme(Osorio et al. 2008, 2011). In adult mice, Runx1 is ex-pressed in the bulge, HG, and HF progenitors. Deletionof Runx1 in the skin epidermis during embryonic devel-opment results in bulge SCs that fail to actively proliferateduring telogen-to-anagen transition, leading to delayed HFregeneration (Osorio et al. 2008). In contrast, Runx1 ex-pression in the embryonic mesenchyme is essential for HFdifferentiation and long-term maintenance of bulge SCs(Osorio et al. 2011).

4.3 Fine-Tuning of Epidermal Differentiationand Homeostasis by Epigenetic Controls

Ablation of microRNA in the epidermis by conditionaldeletion of Dicer or DGDR8 leads to defects in epidermaldifferentiation, such as evaginating HF, increased apopto-sis, impaired barrier function, and neonatal lethality (Andlet al. 2006; Yi et al. 2006, 2009). The expression of micro-RNA is spatiotemporally regulated in the epidermis. Forinstance, the miR-200 and miR-19/miR-20 families arepreferentially expressed in the IFE, whereas the miR-199family is present exclusively in the HF and miR-125b ishighly expressed in HF SCs (Yi et al. 2006; Zhang et al.2011). Moreover, miR-125b is expressed in HF SCs butdramatically down-regulated in their early progeny, where-as sustained miR-125b expression resulted in a reversible

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delay in the transition from SCs to committed progenitors,concomitant with thickened IFE, enlarged SG, and failureto form hair (Zhang et al. 2011). Furthermore, miR-203promotes differentiation and suppresses stemness in theIFE through direct repression of p63. In vitro and in vivoexperiments showed that loss of function of miR-203 in-duces proliferation in the suprabasal cells, whereas gain offunction resulted in exit of the cell cycle and loss of colony-forming potential (Lena et al. 2008; Yi et al. 2008).

Histone methylation is crucial for the epidermal differ-entiation and stratification. Histone deacetylase (HDAC)1/2 directly mediate the repressive functions of p63, par-tially by deacetylating p53, which opposes p63 functions.Double conditional knockout mice for HDAC1/2 havethin, smooth skin and failure for limb-digit separationand eyelid fusion (LeBoeuf et al. 2010). Small interferingRNA–mediated deletion of JMJD3, an H3K27me3 de-methylase in organotypic human epidermal cultures, pre-vented epidermal differentiation, whereas conversely, JMJD3overexpression led to premature differentiation (Sen et al.2008). Whereas conditional deletion in the epidermis ofthe polycomb complex protein Ezh2 alone leads to rathermild phenotype (Ezhkova et al. 2009), conditional deletionof both EZH1/2 differentially affects HF and IFE. HF mor-phogenesis and maintenance are compromised, exhibit-ing low proliferation and increased apoptosis that is atleast partially mediated by ectopic activation of theInk4a/Inkb/Arf locus. Conversely, upon EZH1/2 deletion,IFE exhibits hyperproliferation (Ezhkova et al. 2011). DNAmethyltransferase 1 (DNMT1) and ubiquitinlike, contain-ing PHD and RING finger domains-1 (UHRF1) are ex-pressed in the basal layer of the epidermis and are down-regulated during differentiation. DNMT1 depletion usingshort hairpin RNA led to premature differentiation of theprogenitors and progressive tissue loss in human skin re-generation studies, demonstrating the essential role ofDNMT1 for epidermal self-renewal (Sen et al. 2010).

5 GENOMIC MAINTENANCE IN EPIDERMALSTEM CELLS

The DNA integrity of all cells, including SCs, is continu-ously endangered by genotoxic assaults coming from thesurrounding environment, such as UV radiation and by-products of the cellular metabolism such as radical oxygenspecies (Sancar et al. 2004). It has been estimated that eachcell undergoes as many as 105 spontaneous DNA lesionsper day (Lindahl 1993). As SCs reside and self-renew in theadult tissues over a lifetime, unrepaired DNA damage maybe transmitted to their progeny, leading to precancerousmutations or compromising SC fitness and leading to aging(Blanpain et al. 2011; Mandal et al. 2011). Mutations in

components of DNA repair pathways can lead to prematureaging syndromes, such as xeroderma pigmentosum, Cock-ayne syndrome, and trichothiodystrophy, which are char-acterized by premature aging, neurodegeneration, andincreased incidence of skin cancer (Hoeijmakers 2009).Because of the high cellular turnover of the skin, epidermalSCs have to divide many times throughout life, and thus areat risk of accumulating errors during DNA replication. Ithas been proposed that SCs may undergo asymmetric di-visions during which the SC retains the older (“immortal”)DNA strand and the committed daughter cell inherits thenewest synthesized DNA strand (Cairns 1975). Double-la-beling pulse-chase experiments (Sotiropoulou et al. 2008)and chromatin labeling with single uridine labeling (Wagh-mare et al. 2008) provided evidence that in vivo HF SCssegregate their chromosomes randomly. Consistent withthe skin barrier function, epidermal SCs are continuouslyassaulted by UV irradiation. Studies in human and mousehave shown that the proliferating basal cells of the IFE aremore sensitive to UV irradiation compared with the moredifferentiated suprabasal cells (reviewed in Blanpain et al.2011). Upon UV irradiation the IFE basal cells exhibit sus-tained p53 accumulation and higher apoptosis rates, con-comitant with an inverse gradient of Nrf2 expression,which controls the expression of oxidative stress regulators(Schafer et al. 2010).

Ex vivo studies of human epidermal SCs have shownthat they are more resistant to ionizing radiation, possiblybecause of an autocrine/paracrine FGF2-mediated in-crease in DNA repair activity (Rachidi et al. 2007; Har-fouche et al. 2010). HF SCs are also more resistant toDNA damage-induced cell death compared with theirmore mature counterparts (Sotiropoulou et al. 2010).Higher expression of the antiapoptotic factor Bcl-2 andthe shorter duration of DNA damage response in HFs be-cause of accelerated nonhomologous end-joining (NHEJ)DNA repair activity contribute to the increased resistanceof HF SCs to DNA damage-induced cell death (Fig. 4). Theimportance of controlled DNA damage response in HFSCs is illustrated by the SC exhaustion and progressivealopecia in mice lacking Atr (Ruzankina et al. 2007) andthe SC senescence and premature skin aging in mice lackingMdm2, a negative regulator of p53 (Gannon et al. 2011).Because NHEJ is an error-prone DNA repair mechanism,this mechanism could be a double-edged sword promot-ing immediate cell survival at the expense of long-termgenomic integrity, potentially leading to the accumulationof precancerous mutations. Indeed, mice deficient forNHEJ and the antiapoptotic factor Bcl-XL are more resis-tant to chemical-induced carcinogenesis because of in-creased apoptosis and elimination of mutated SCs (Kempet al. 1999; Kim et al. 2009b).

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6 EPIDERMAL STEM CELLS AND CANCERINITIATION

BCC and SCC are the most frequent skin cancers and themost common neoplasias in humans, arising from sun-ex-posed regions of the skin. Because SCs exist and proliferatefor extended periods in adult tissue, they present, in theory,a higher chance of accumulating the necessary mutations toinduce cancer formation (Pardal et al. 2003; Clarke andFuller 2006). However, for most cancers, the precise identi-fication of the tumor-initiating cells remains elusive.

6.1 Basal Cell Carcinoma

BCC is a slow-growing and locally invasive cancer, withmore than a million new cases each year worldwide, andis believed to arise from the HF because of its histologicaland biochemical resemblances to HFs (Owens and Watt

2003; Perez-Losada and Balmain 2003). All BCCs presentconstitutive hedgehog (HH) activation, loss of heterozy-gosity of Patched being the most common genetic alter-ation, followed by constitutively active mutations inSmoothened (Hahn et al. 1996; Johnson et al. 1996; Xieet al. 1998; Epstein 2008). Mouse models for BCC, eitherby Patched loss of function or by gain-of-function mu-tations of Shh, SmoM2, and their downstream transcrip-tion factors (Gli1 and Gli2) (Dahmane et al. 1997; Fanet al. 1997; Grachtchouk et al. 2000, 2003; Mao et al.2006), give rise to skin lesions that closely resemble humanBCC.

Conditional expression of the SmoM2 oncogene showedthat during physiological conditions, bulge SCs and theirprogeny are highly resistant to SmoM2-induced BCC de-velopment (Fig. 5) (Youssef et al. 2010; Wong and Reiter2011). Using clonal analysis of oncogene-targeted cells,

DNA-PK

DNAdamage

DNAdamage response

Proapoptotic genes(Puma, Noxa, Bax)

Cell death

ProsurvivalBcl-2

DNA repair

Stem cellmaintenance

p53

H2AX

ATMChk2

Figure 4. Mechanisms ensuring genomic integrity in skin stem cells. Upon DNA damage, hair follicle bulge stem cellsinitiate the DNA damage response pathway, but because of higher levels of antiapoptotic Bcl-2 and DNA-PK (redarrows), they exhibit lower DNA damage-induced cell death, accelerated DNA repair, faster attenuation of p53stabilization, and inhibition of apoptosis.

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Kass Youssef and colleagues showed that more than 90% ofSmoM2-induced BCCs arise from long-lived progenitorsresiding in the IFE, the rest arising from the upper infun-dibulum (Youssef et al. 2010). Bulge SCs generate hyper-plastic or dysplastic lesions upon SmoM2 expression, whichdo not progress into invasive cancer (Fig. 5) (Youssef et al.2010). However, when SmoM2-expressing bulge SCs arestimulated to migrate into the IFE following wounding,bulge SC progeny are competent to progress into invasiveBCC (Kasper et al. 2011; Wong and Reiter 2011). Using apharmacological trick to specifically delete Patched-1 in theIFE and the infundibulum, these cells were also shown to becompetent to initiate BCC formation (Adolphe et al. 2006).Patched-1 deletion in bulge SC and its progeny using

Lgr5CREER led to BCC at low frequency, indicating thatbulge SCs are not completely resistant to HH-mediatedBCC development, and their competence is greatly en-hanced upon wounding. This indicates that either the mi-croenvironment of IFE is more permissive or that followingmigration to the IFE (Kasper et al. 2011) bulge SCs arereprogrammed into an IFE fate, which is required to allowthe oncogenic program dictated by HH signaling. Geneticlineage-tracing experiments followed by ionizing radiationsuggested that bulge SCs could be the cellular origin ofionizing radiation-induced BCC (Wang et al. 2011). How-ever, the infundibulum and IFE-ectopic activity of theK15CREPR, the K15 expression in BCC (Youssef et al.2010), and the leakiness of K15CREPR (Lapouge et al.

Squamous cell carcinomaBasal cell carcinoma

(SMO-M2)

Total basal epidermal cells (K14CreER,K6Cre+RA-treatment)

Transient amplifying cells(ShhCreER)

HF Bulge stem cells(K19CreER, K15CrePR, Lgr5CreER)

IFE cells(InvCreER)

(Patched1f/f) (KRasLSL-G12D) (KRasLSL-G12D/p53f/f)

No effect No effectNo effect

Dysplasia

BCC SCC

SCC

Papilloma

Papilloma

Papilloma

Nowound

Pluswound

BCC+++

BCC+++

BCC+

Suprabasal IFE cells(K10-HRas) Papillomas at sites of wounding

HF cells(ΔK5-HRas) Papillomas and SCC

Nowound

Pluswound

BCC++

Figure 5. Cells at the origin of the most common types of skin cancer. Activation of Hedgehog signaling in differentcellular compartments of the epidermis using mice conditionally expressing constitutively active Smoothenedmutant (SmoM2) showed that BCC does not arise by hair follicle stem cells as previously believed, but fromlong-term resident progenitor cells of the interfollicular epidermis and the upper infundibulum. Likewise, usingthe same approach and mice conditionally expressing a constitutively active KRas mutant (G12D), it was shown thatsquamous tumors can be generated by both interfollicular epidermis and hair follicle stem cells, but not theirimmediate progeny, whereas additional mutations have to occur to progress to the malignant state.

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2011) may also explain the apparent bulge SC origin ofBCC in this model.

The primary cilium represents a small membrane or-ganelle that plays an important role in regulating HH sig-naling (Goetz and Anderson 2010). Deletion of Kif3a orItf88, critical for cilia formation, results in hyperprolifera-tive skin lesions and excessive migration of bulge SCs to theIFE (Croyle et al. 2011). Human BCCs are also ciliated, anddeletion of Kif3a or Itf88 blocked SmoM2-induced tumor-igenesis but enhanced Gli2-mediated BCC formation(Wong et al. 2009), suggesting that cilia can either poten-tiate or inhibit tumorigenesis depending on the oncogeniccontext.

Different HH target genes such as Sox9 and Wnt ligandsand receptors are expressed in mouse and human BCC(Vidal et al. 2005; Yang et al. 2008), and blocking Wntsignaling by Dkk1 overexpression inhibits SmoM2-inducedtumorigenesis during skin morphogenesis (Yang et al.2008). The exact role of Wnt signaling and Sox9 express-ion during BCC development and whether these signalingpathways control similar functions during HF morphogen-esis and tumor development remain to be determined.

6.2 Squamous Cell Carcinoma

SCC is the second-most-frequent skin cancer, with morethan 500,000 patients per year worldwide (Alam and Rat-ner 2001). The most extensively used mouse model for skinSCC is a multistage, chemically induced carcinogenesis(Kemp 2005), in which mice are treated first with a muta-gen (the 9,10-dimethyl-1,2-benzanthracene, DMBA) andthen with a drug that stimulates epidermal proliferation,such as 12-O-tetradecanoyl phorbol-13-acetate (TPA).During TPA treatment, benign tumors (papillomas) arise,probably as a consequence of additional mutations, someof which progress into invasive SCC. This model selectscells with active mutations in the Ras pathway. Althoughthe most common form of Ras mutations found in papil-lomas occurred in the HRas gene (Quintanilla et al. 1986),mutations in the KRas gene have also been reported (vander Schroeff et al. 1990; Sutter et al. 1993; Spencer et al.1995).

SCC often present signs of squamous differentiation,suggesting that SCC may originate from cells that naturallyundergo squamous differentiation such as the IFE (Owensand Watt 2003). TPA administration stimulates papillomaformation even when started a year after DMBA treatment,suggesting that the initial mutation arises in long-livedSCs (Morris et al. 1986, 1997; Morris 2000). The demon-stration that tumors can form, although at lower frequency,when the IFE is removed by dermabrasion after DMBAand repaired by HF cells suggests that cancer-initiating cells

may reside in the IFE as well as in HFs (Argyris and Slaga1981). However, some studies suggest that oncogenic mu-tations could also occur in differentiated cells of the epi-dermis (Owens et al. 2003). Suprabasal expression of a6b4integrins increases papilloma formation following DMBA/TPA treatment and expression of active MEK in suprabasalcells enhances papilloma formation following wounding.The promotion of tumorigenesis by differentiated cells hasbeen proposed to be mediated through an increase in in-flammation (Arwert et al. 2010). In addition, transgenicmice expressing a mutated form of HRas in IFE suprabasalcells under the promoter K10 develop papillomas at sites ofwounding (Bailleul et al. 1990). Transgenic mice expressingHRas under the control of a truncated form of the K5promoter are preferentially expressed in the HF of adultmice, and develop papillomas and SCC (Brown et al.1998). Mosaic activation of a constitutively active mutantof KRas (KRasLSL-G12D) mimicking the natural occurrenceof sporadic tumors in different compartments of the skinepidermis revealed the SCC-initiating cells (Fig. 5) (La-pouge et al. 2011; White et al. 2011). Papilloma forma-tion was observed upon KRasG12D expression in bulgeSCs and their progeny, but not in HF transit-amplifyingcells. KRasG12D expression in the IFE-induced hyperprolif-eration and hyperplasia, sometimes progressing into pap-illomas, indicating that cells of the IFE are competent todevelop papillomas (Lapouge et al. 2011). The cellular or-igin of DMBA/TPA-induced papilloma and SCC has notbeen determined yet, but the absence of tumors in DMBA/TPA-treated CD34-null mice (Trempus et al. 2007) and theresistance to DMBA/TPA-induced tumor formation inmice with Stat3 deletion in bulge SCs and their progeny(Chan et al. 2004; Kim et al. 2009a) indicate that bulge SCsprobably participate in papilloma formation.

No sign of malignant transformation was observedupon oncogenic KRas expression alone, suggesting thatother genetic events are required to induce the develop-ment of invasive SCC (Fig. 5). Combined p53 deletionand KRasG12D expression in the basal layer of the epidermisand in bulge SCs rapidly induced SCC, suggesting thatat least two genetic hits are required to mediate SCC initi-ation in the context of oncogenic Ras (Fig. 5) (Lapougeet al. 2011; White et al. 2011). Whereas p53 loss of func-tion has been associated with malignant progression (Ow-ens and Watt 2003; Perez-Losada and Balmain 2003), p53gain-of-function mutation has been shown to increase tu-mor initiation and progression (Caulin et al. 2007).More studies are needed to better understand SCC initia-tion, which genes control the renewal and malignant pro-gression of oncogene-targeted cells, what is the role of thetumor microenvironment, and how p53 controls malig-nant progression.

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7 THERAPEUTIC APPLICATIONS OF SKINSTEM/PROGENITOR CELLS

Skin stem cells are already being used for patients withsevere burns, as well as several skin disorders (Fig. 6). Sincethe first demonstration by Green and colleagues in 1975that skin keratinocytes can be grown in vitro to form co-hesive sheets of cells (Rheinwald and Green 1975), numer-ous improvements have been proposed, and culture of asmall skin biopsy can now provide sufficient epidermalcells for extensive autograft. Currently, the use of culturedepidermal grafts is hampered by the length of the culturetime, the type of burn surface influencing the graft adher-ence, and the high cost. Bioengineered skin equivalentshave been proposed, such as the use of collagen-based ma-trices or human plasma as dermal scaffold, but their use inclinic is still limited (Alonso and Fuchs 2003). The use ofcryopreserved allogeneic skin grafts, which are replaced bythe epithelial autografts when ready, provides the best re-sults in terms of functional and esthetic results, but unfor-tunately is more expensive and requires special facilities(Atiyeh and Costagliola 2007).

Cultured skin SCs also hold great promise for the treat-ment of genetic skin diseases (Fig. 6). Preclinical studieshave suggested that skin SCs can be used for the treatmentof disorders such as epidermolysis bullosa, an inheriteddeficiency of components of hemidesmosomes. Keratino-cytes have been isolated from epidermolysis bullosa pa-tients, the deficiency in laminin-5 has been corrected invitro, and the cells have been grafted back to the patients,resulting in stable correction of the disease in the trans-planted site (Mavilio et al. 2006; Siprashvili et al. 2010).

In conclusion, although cell therapy based on skin SCsis already currently used in clinical practice, there are spe-cific hurdles that need to be overcome before it is moregenerally applied, such as improving the culture conditionsso as to obtain larger numbers of cells in less time, as well as

understanding why a fraction of skin transplants do notengraft. It will be a great improvement for the comfort ofpatients to be able to reconstruct the skin appendages, inparticular the sweat glands, but also the hair follicles. SkinSCs may also hold promise to treat additional disorders,and preclinical studies should evaluate the types of diseaseand the safety of these therapeutic approaches.

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Skin biopsy

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